Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids.

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a "plasma membrane on a chip," also known as a supported lipid bilayer. Here, we create the "plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein-protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein-protein and protein-lipid interactions in a convenient, cell-free platform.

Indole-3-glycerolphosphate synthase, a branchpoint for the biosynthesis of tryptophan, indole, and benzoxazinoids in maize.

The maize (Zea mays) genome encodes three indole-3-glycerolphosphate synthase enzymes (IGPS1, 2, and 3) catalyzing the conversion of 1-(2-carboxyphenylamino)-l-deoxyribulose-5-phosphate to indole-3-glycerolphosphate. Three further maize enzymes (BX1, benzoxazinoneless 1; TSA, tryptophan synthase alpha subunit; and IGL, indole glycerolphosphate lyase) convert indole-3-glycerolphosphate to indole, which is released as a volatile defense signaling compound and also serves as a precursor for the biosynthesis of tryptophan and defense-related benzoxazinoids. Phylogenetic analyses showed that IGPS2 is similar to enzymes found in both monocots and dicots, whereas maize IGPS1 and IGPS3 are in monocot-specific clades. Fusions of yellow fluorescent protein with maize IGPS enzymes and indole-3-glycerolphosphate lyases were all localized in chloroplasts. In bimolecular fluorescence complementation assays, IGPS1 interacted strongly with BX1 and IGL, IGPS2 interacted primarily with TSA, and IGPS3 interacted equally with all three indole-3-glycerolphosphate lyases. Whereas IGPS1 and IGPS3 expression was induced by insect feeding, IGPS2 expression was not. Transposon insertions in IGPS1 and IGPS3 reduced the abundance of both benzoxazinoids and free indole. Spodoptera exigua (beet armyworm) larvae show improved growth on igps1 mutant maize plants. Together, these results suggest that IGPS1 and IGPS3 function mainly in the biosynthesis of defensive metabolites, whereas IGPS2 may be involved in the biosynthesis of tryptophan. This metabolic channeling is similar to, though less exclusive than, that proposed for the three maize indole-3-glycerolphosphate lyases.

Genome sequence of the corn leaf aphid (Rhopalosiphum maidis Fitch).

BACKGROUND: The corn leaf aphid (Rhopalosiphum maidis Fitch) is the most economically damaging aphid pest on maize (Zea mays), one of the world's most important grain crops. In addition to causing direct damage by removing photoassimilates, R. maidis transmits several destructive maize viruses, including maize yellow dwarf virus, barley yellow dwarf virus, sugarcane mosaic virus, and cucumber mosaic virus. FINDINGS: The genome of a parthenogenetically reproducing R. maidis clone was assembled with a combination of Pacific Biosciences (207-fold coverage) and Illumina (83-fold coverage) sequencing. The 689 assembled contigs, which have an N50 size of 9.0 megabases (Mb) and a low level of heterozygosity, were clustered using Phase Genomics Hi-C interaction maps. Consistent with the commonly observed 2n = 8 karyotype of R. maidis, most of the contigs (473 spanning 321 Mb) were successfully oriented into 4 scaffolds. The genome assembly captured the full length of 95.8% of the core eukaryotic genes, indicating that it is highly complete. Repetitive sequences accounted for 21.2% of the assembly, and a total of 17,629 protein-coding genes were predicted with integrated evidence from ab initio and homology-based gene predictions and transcriptome sequences generated with both Pacific Biosciences and Illumina. An analysis of likely horizontally transferred genes identified 2 from bacteria, 7 from fungi, 2 from protozoa, and 9 from algae. Repeat elements, transposons, and genes encoding likely detoxification enzymes (cytochrome P450s, glutathione S-transferases, carboxylesterases, uridine diphosphate-glucosyltransferases, and ABC transporters) were identified in the genome sequence. Other than Buchnera aphidicola (642,929 base pairs, 602 genes), no endosymbiont bacteria were found in R. maidis. CONCLUSIONS: A high-quality R. maidis genome was assembled at the chromosome level. This genome sequence will enable further research related to ecological interactions, virus transmission, pesticide resistance, and other aspects of R. maidis biology. It also serves as a valuable resource for comparative investigation of other aphid species.

Metabolome-Scale Genome-Wide Association Studies Reveal Chemical Diversity and Genetic Control of Maize Specialized Metabolites.

Cultivated maize (Zea mays) has retained much of the genetic diversity of its wild ancestors. Here, we performed nontargeted liquid chromatography-mass spectrometry metabolomics to analyze the metabolomes of the 282 maize inbred lines in the Goodman Diversity Panel. This analysis identified a bimodal distribution of foliar metabolites. Although 15% of the detected mass features were present in >90% of the inbred lines, the majority were found in <50% of the samples. Whereas leaf bases and tips were differentiated by flavonoid abundance, maize varieties (stiff-stalk, nonstiff-stalk, tropical, sweet maize, and popcorn) showed differential accumulation of benzoxazinoid metabolites. Genome-wide association studies (GWAS), performed for 3,991 mass features from the leaf tips and leaf bases, showed that 90% have multiple significantly associated loci scattered across the genome. Several quantitative trait locus hotspots in the maize genome regulate the abundance of multiple, often structurally related mass features. The utility of maize metabolite GWAS was demonstrated by confirming known benzoxazinoid biosynthesis genes, as well as by mapping isomeric variation in the accumulation of phenylpropanoid hydroxycitric acid esters to a single linkage block in a citrate synthase-like gene. Similar to gene expression databases, this metabolomic GWAS data set constitutes an important public resource for linking maize metabolites with biosynthetic and regulatory genes.

The Developing Regorafenib Eye drops for neovascular Age-related Macular degeneration (DREAM) study: an open-label phase II trial.

AIMS: This programme investigated topical regorafenib, a multikinase inhibitor, in patients with neovascular age-related macular degeneration (nAMD). METHODS: Topical regorafenib was investigated in an open-label, phase IIa/b study in which patients with choroidal neovascularization (CNV) secondary to nAMD received regorafenib (25 µl, 30 mg ml-1 ) three times a day for 12 weeks. The primary endpoint of the phase II/a/b study was mean change in best-corrected visual acuity (BCVA) from baseline to weeks 4 and 12. RESULTS: In nAMD patients (N = 51), mean changes in BCVA were +1.2 [90% confidence interval (CI) -0.61, 2.97] and -2.4 (90% CI -4.18, -0.54) letters at weeks 4 and 12, respectively. Ocular treatment-emergent adverse events (TEAEs) (study eye) were reported in 21 patients by week 12. There was one serious ocular TEAE (visual acuity reduced) that was not drug related. Twenty patients required rescue (intravitreal ranibizumab). CONCLUSIONS: The programme was terminated after phase IIa ended because efficacy was lower than with current nAMD treatments. According to elaborate post hoc analyses, the most likely reason was insufficient exposure in the target compartment (back of the eye).

Beyond Defense: Multiple Functions of Benzoxazinoids in Maize Metabolism.

Benzoxazinoids are a class of indole-derived plant metabolites that function in defense against numerous pests and pathogens. Due to their abundance in maize (Zea mays) and other important cereal crops, benzoxazinoids have been the subject of extensive research for >50 years. Whereas benzoxazinoids can account for 1% or more of the dry weight in young seedlings constitutively, their accumulation in older plants is induced locally by pest and pathogen attack. Although the biosynthetic pathways for most maize benzoxazinoids have been identified, unanswered questions remain about the developmental and defense-induced regulation of benzoxazinoid metabolism. Recent research shows that, in addition to their central role in the maize chemical defense repertoire, benzoxazinoids may have important functions in regulating other defense responses, flowering time, auxin metabolism, iron uptake and perhaps aluminum tolerance. Investigation of natural variation in maize benzoxazinoid accumulation, which is greatly facilitated by recent genomics advances, will have a major impact in this research area by leading to the discovery of previously unknown genes and functions of benzoxazinoid metabolism.

A maize landrace that emits defense volatiles in response to herbivore eggs possesses a strongly inducible terpene synthase gene.

Maize (Zea mays) emits volatile terpenes in response to insect feeding and egg deposition to defend itself against harmful pests. However, maize cultivars differ strongly in their ability to produce the defense signal. To further understand the agroecological role and underlying genetic mechanisms for variation in terpene emission among maize cultivars, we studied the production of an important signaling component (E)-caryophyllene in a South American maize landrace Braz1006 possessing stemborer Chilo partellus egg inducible defense trait, in comparison with the European maize line Delprim and North American inbred line B73. The (E)-caryophyllene production level and transcript abundance of TPS23, terpene synthase responsible for (E)-caryophyllene formation, were compared between Braz1006, Delprim, and B73 after mimicked herbivory. Braz1006-TPS23 was heterologously expressed in E. coli, and amino acid sequences were determined. Furthermore, electrophysiological and behavioral responses of a key parasitic wasp Cotesia sesamiae to C. partellus egg-induced Braz1006 volatiles were determined using coupled gas chromatography electroantennography and olfactometer bioassay studies. After elicitor treatment, Braz1006 released eightfold higher (E)-caryophyllene than Delprim, whereas no (E)-caryophyllene was detected in B73. The superior (E)-caryophyllene production by Braz1006 was positively correlated with high transcript levels of TPS23 in the landrace compared to Delprim. TPS23 alleles from Braz1006 showed dissimilarities at different sequence positions with Delprim and B73 and encodes an active enzyme. Cotesia sesamiae was attracted to egg-induced volatiles from Braz1006 and synthetic (E)-caryophyllene. The variation in (E)-caryophyllene emission between Braz1006 and Delprim is positively correlated with induced levels of TPS23 transcripts. The enhanced TPS23 activity and corresponding (E)-caryophyllene production by the maize landrace could be attributed to the differences in amino acid sequence with the other maize lines. This study suggested that the same analogous genes could have contrasting expression patterns in different maize genetic backgrounds. The current findings provide valuable insight not only into genetic mechanisms underlying variation in defense signal production but also the prospect of introgressing the novel defense traits into elite maize varieties for effective and ecologically sound protection of crops against damaging insect pests.

Characterization of Biosynthetic Pathways for the Production of the Volatile Homoterpenes DMNT and TMTT in Zea mays.

Plant volatiles not only have multiple defense functions against herbivores, fungi, and bacteria, but also have been implicated in signaling within the plant and toward other organisms. Elucidating the function of individual plant volatiles will require more knowledge of their biosynthesis and regulation in response to external stimuli. By exploiting the variation of herbivore-induced volatiles among 26 maize (Zea mays) inbred lines, we conducted a nested association mapping and genome-wide association study (GWAS) to identify a set of quantitative trait loci (QTLs) for investigating the pathways of volatile terpene production. The most significant identified QTL affects the emission of (E)-nerolidol, linalool, and the two homoterpenes (E)-3,8-dimethyl-1,4,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). GWAS associated a single nucleotide polymorphism in the promoter of the gene encoding the terpene synthase TPS2 with this QTL Biochemical characterization of TPS2 verified that this plastid-localized enzyme forms linalool, (E)-nerolidol, and (E,E)-geranyllinalool. The subsequent conversion of (E)-nerolidol into DMNT maps to a P450 monooxygenase, CYP92C5, which is capable of converting nerolidol into DMNT by oxidative degradation. A QTL influencing TMTT accumulation corresponds to a similar monooxygenase, CYP92C6, which is specific for the conversion of (E,E)-geranyllinalool to TMTT The DMNT biosynthetic pathway and both monooxygenases are distinct from those previously characterized for DMNT and TMTT synthesis in Arabidopsis thaliana, suggesting independent evolution of these enzymatic activities.

Dynamic Maize Responses to Aphid Feeding Are Revealed by a Time Series of Transcriptomic and Metabolomic Assays.

As a response to insect attack, maize (Zea mays) has inducible defenses that involve large changes in gene expression and metabolism. Piercing/sucking insects such as corn leaf aphid (Rhopalosiphum maidis) cause direct damage by acquiring phloem nutrients as well as indirect damage through the transmission of plant viruses. To elucidate the metabolic processes and gene expression changes involved in maize responses to aphid attack, leaves of inbred line B73 were infested with corn leaf aphids for 2 to 96 h. Analysis of infested maize leaves showed two distinct response phases, with the most significant transcriptional and metabolic changes occurring in the first few hours after the initiation of aphid feeding. After 4 d, both gene expression and metabolite profiles of aphid-infested maize reverted to being more similar to those of control plants. Although there was a predominant effect of salicylic acid regulation, gene expression changes also indicated prolonged induction of oxylipins, although not necessarily jasmonic acid, in aphid-infested maize. The role of specific metabolic pathways was confirmed using Dissociator transposon insertions in maize inbred line W22. Mutations in three benzoxazinoid biosynthesis genes, Bx1, Bx2, and Bx6, increased aphid reproduction. In contrast, progeny production was greatly decreased by a transposon insertion in the single W22 homolog of the previously uncharacterized B73 terpene synthases TPS2 and TPS3. Together, these results show that maize leaves shift to implementation of physical and chemical defenses within hours after the initiation of aphid feeding and that the production of specific metabolites can have major effects in maize-aphid interactions.

A small, differentially regulated family of farnesyl diphosphate synthases in maize (Zea mays) provides farnesyl diphosphate for the biosynthesis of herbivore-induced sesquiterpenes.

MAIN CONCLUSION: Of the three functional FPPS identified in maize, fpps3 is induced by herbivory to produce FDP important for the formation of the volatile sesquiterpenes of plant defense. Sesquiterpenes are not only crucial for the growth and development of a plant but also for its interaction with the environment. The biosynthesis of sesquiterpenes proceeds over farnesyl diphosphate (FDP), which is either used as a substrate for protein prenylation, converted to squalene, or to volatile sesquiterpenes. To elucidate the regulation of sesquiterpene biosynthesis in maize, we identified and characterized the farnesyl diphosphate synthase (FPPS) gene family which consists of three genes. Synteny analysis indicates that fpps2 and fpps3 originate from a genome duplication in an ancient tetraploid ancestor. The three FPPSs encode active enzymes that produce predominantly FDP from the isopentenyl diphosphate and dimethylallyl diphosphate substrates. Only fpps1 and fpps3 are induced by elicitor treatment, but induced fpps1 levels are much lower and only increased to the amounts of fpps3 levels in intact leaves. Elicitor-induced fpps3 levels in leaves increase to more than 15-fold of background levels. In undamaged roots, transcript levels of fpps1 are higher than those of fpps3, but only fpps3 transcripts are induced in response to herbivory by Diabrotica virgifera virgifera. A kinetic of transcript abundance in response to herbivory in leaves provided further evidence that the regulation of fpps3 corresponds to that of tps23, a terpene synthase, that converts FDP to the volatile (E)-ß-caryophyllene. Our study indicates that the differential expression of fpps1 and fpps3 provides maize with FDP for both primary metabolism and terpene-based defenses. The expression of fpps3 seems to coincide with the herbivore-induced emission of volatile sesquiterpenes that were demonstrated to be important defense signals.

Polymeric micelles for parenteral delivery of sagopilone: physicochemical characterization, novel formulation approaches and their toxicity assessment in vitro as well as in vivo.

PURPOSE: The block copolymers PEG(2000)-b-PLA(2200), PEG(2000)-b-PCL(2600) and PEG(5000)-b-PCL(5000) have been currently identified as optimal solubilizing agents for Sagopilone, a poorly water-soluble anticancer drug. In the present study, the stability, formulation feasibility and in vitro as well as in vivo toxicity were evaluated. METHODS: Dispersion media, storage conditions, and dilutions were varied for stability assessment. The critical micelle concentration (CMC) was determined using a fluorescent probe technique. Lyophilizates and polymeric films were investigated as formulation options. Furthermore, the toxicity was studied in vitro and in vivo using HeLa/MaTu cells and a nude mouse model, respectively. RESULTS: A drug-polymer ratio as low as 1:20 (w/w) was sufficient to solubilize Sagopilone effectively and to obtain stable dispersions (24h: drug content >or= 95%). Although the micelles exhibited a similar thermodynamic stability (CMC: 10(-7)-10(-6)M), PEG-b-PCL micelles were kinetically more stable than PEG(2000)-b-PLA(2200) (24h at 37 degrees C: drug content >or= 90% compared to 30%, respectively). Lyophilization of PEG-b-PCL micelles and storage stability of solid drug-loaded PEG(2000)-b-PLA(2200) films (3m, 6 degrees C: drug content of (95.6+/-1.4)%) were demonstrated for the first time. The high antiproliferative activity has been maintained in vitro (IC(50)<1 nM). Carrier-associated side effects have not been observed in vivo and the maximum tolerated dose of micellar Sagopilone was determined to be 6 mg/kg. CONCLUSION: The results of this study indicate that polymeric micelles, especially PEG-b-PCL micelles, offer excellent potential for further preclinical and clinical cancer studies using Sagopilone.

Non-ionic dendritic glycerol-based amphiphiles: novel excipients for the solubilization of poorly water-soluble anticancer drug Sagopilone.

The purpose of this study was to investigate dendritic glycerol-based amphiphiles as novel solubilizers using poorly water-soluble anticancer drug Sagopilone. The effect of different core structures on the solubilization, formulation stability, and cytotoxicity using human umbilical vein endothelial cells (HUVECs) were investigated and compared to standard excipients. Structurally, all amphiphiles were composed of 2nd generation polyglycerol (PG[G2]) as the hydrophilic part and a single C(18)-chain (PG[G2]-C(18)), a C(18)-chain coupled by a diaromatic spacer (PG[G2]-DiAr-C(18)), a C(18)-chain with a naphthyl or bisphenyl end group (PG[G2]-C(18)-Naph/-BiP), or two C(18)-chains (PG[G2]-(C(18))(2)) as the hydrophobic part. They formed small (7-10 nm), monodisperse (PDI 0.04-0.20) micelles with the exception of PG[G2]-(C(18))(2). The amphiphiles revealed a 2-3-fold higher solubilization of Sagopilone than Cremophor ELP and polysorbate 80 independent of the core structure. PG[G2]-DiAr-C(18) exhibited the highest solubilization capacity (56.7+/-1.3 mg/g) compared to Cremophor ELP (18.5+/-0.1 mg/g). The micellar dispersions were stable in drug content over 3 days (> or = 97%). In contrast to polysorbate 80, dilutions did not show any precipitation after 3 days at 37 degrees C (remaining drug content: > 95%). They did not induce significant cytotoxicity at a concentration of 0.01 g/L after 24 h, and PG[G2]-C(18)-Naph was the least cytotoxic structure after 72 h with values comparable to Cremophor ELP and polysorbate 80. Overall, these amphiphiles possess superior solubilization properties compared to standard excipients used in parenteral formulations with an excellent formulation stability profile and comparable cytotoxicity.

Solubilization of sagopilone, a poorly water-soluble anticancer drug, using polymeric micelles for parenteral delivery.

Polymeric micelles were studied as a drug delivery system for Sagopilone, a poorly water-soluble anticancer drug, with respect to passive tumour targeting. Poly(ethylene glycol)-b-Poly(lactide) (PEG-b-PLA) and Poly(ethylene glycol)-b-Poly(epsilon-caprolactone) (PEG-b-PCL) were investigated to identify suitable copolymers and to assess the predictive value of solubility parameters. The impact of copolymer compositions (different hydrophobic/hydrophilic-ratios (w/w) from 0.3 to 1.3) and the preparation method (sonication; film formation) on the solubilization efficiency, size characteristics and micelle stability were studied. Thermal analysis was used to determine the apparent solid-state solubility. PEG(2000)-b-PLA(2200), PEG(2000)-b-PCL(2600) and PEG(5000)-b-PCL(5000) were identified as the most suitable delivery systems for Sagopilone. They exhibited efficient solubilization (> or =70%) yielding small (<100 nm), monodisperse, and spherical micelles. (80+/-12), (93+/-0.4) and (96+/-6)% of the drug still remained solubilized after 24h, respectively. Calculated solubility parameters were not predictive since they showed a reversed order of preference relative to experimental data. High solubilization after film hydration was accompanied with a 'supersaturation'. The reason for this well-known effect and the solubilization of Sagopilone within the block copolymer was elucidated by the evidence of glass solutions exceeding the solubilization capacity of the corresponding micelles. Overall, micellar drug delivery systems for Sagopilone were identified offering the potential for an improved cancer therapy.